Prevention of acute liver injury by suppressing plasma kallikrein-dependent activation of latent TGF-ß.

Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor ß (TGF-ß) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-ß is activated partly through proteolytic cleavage by a serine protease plasma kallikrein (PLK) between the R58 and L59 residues of its propeptide region. Recently, we developed a specific monoclonal antibody to detect the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs) that reflect PLK-dependent TGF-ß activation. This study aimed to explore the potential roles of PLK-dependent TGF-ß activation in the pathogenesis of ALI. We established a mouse ALI model via the injection of anti-Fas antibodies (Jo2) and observed increases in the TGF-ß1 mRNA level, Smad3 phosphorylation, TUNEL-positive apoptotic hepatocytes and R58-positive cells in the liver tissues of Jo2-treated mice. The R58 LAP-DPs were observed in/around F4/80-positive macrophages, while macrophage depletion with clodronate liposomes partly alleviated the Jo2-induced liver injury. Blocking PLK-dependent TGF-ß activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-ß receptor-mediated signaling pathway using SB431542 significantly prevented Jo2-induced hepatic apoptosis and mortality. Furthermore, similar phenomena were observed in the mouse model of ALI with the administration of acetaminophen (APAP). In summary, R58 LAP-DPs reflecting PLK-dependent TGF-ß activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-ß activation has potential as a therapeutic strategy for ALI.

RNA Interference Directed against the Transglutaminase Gene Triggers Dysbiosis of Gut Microbiota in Drosophila.

We recently reported that transglutaminase (TG) suppresses immune deficiency pathway-controlled antimicrobial peptides (IMD-AMPs), thereby conferring immune tolerance to gut microbes, and that RNAi of the TG gene in flies decreases the lifespan compared with non-TG-RNAi flies. Here, analysis of the bacterial composition of the Drosophila gut by next-generation sequencing revealed that gut microbiota comprising one dominant genus of Acetobacter in non-TG-RNAi flies was shifted to that comprising two dominant genera of Acetobacter and Providencia in TG-RNAi flies. Four bacterial strains, including Acetobacter persici SK1 and Acetobacter indonesiensis SK2, Lactobacillus pentosus SK3, and Providencia rettgeri SK4, were isolated from the midgut of TG-RNAi flies. SK1 exhibited the highest resistance to the IMD-AMPs Cecropin A1 and Diptericin among the isolated bacteria. In contrast, SK4 exhibited considerably lower resistance against Cecropin A1, whereas SK4 exhibited high resistance to hypochlorous acid. The resistance of strains SK1-4 against IMD-AMPs in in vitro assays could not explain the shift of the microbiota in the gut of TG-RNAi flies. The lifespan was reduced in gnotobiotic flies that ingested both SK4 and SK1, concomitant with the production of reactive oxygen species and apoptosis in the midgut, whereas the survival rate was not altered in gnotobiotic flies that mono-ingested either SK4 or SK1. Interestingly, significant amounts of reactive oxygen species were detected in the midgut of gnotobiotic flies that ingested SK4 and SK2, concomitant with no significant apoptosis in the midgut. In gnotobiotic flies that co-ingested SK4 and SK1, an additional unknown factor(s) may be required to cause midgut apoptosis.

Transglutaminase-catalyzed protein-protein cross-linking suppresses the activity of the NF-κB-like transcription factor relish.

Cross-linking of proteins by mammalian transglutaminases (TGs) plays important roles in physiological phenomena such as blood coagulation and skin formation. We show that Drosophila TG suppressed innate immune signaling in the gut. RNA interference (RNAi) directed against TG reduced the life span of flies reared under conventional nonsterile conditions but not of those raised under germ-free conditions. In conventionally reared flies, TG RNAi enhanced the expression of genes encoding antimicrobial peptides in the immune deficiency (IMD) pathway. Wild-type flies that ingested gut lysates prepared from conventionally reared TG RNAi-treated flies had shorter life spans. In conventionally reared flies, TG RNAi triggered apoptosis in the gut and induced the nuclear translocation of Relish, the NF-κB (nuclear factor κB)-like transcription factor of the IMD pathway. Wild-type flies that ingested synthetic amine donors, which inhibit the TG-catalyzed protein-protein cross-linking reaction, showed nuclear translocation of Relish and enhanced expression of genes encoding IMD-controlled antimicrobial peptide genes in the gut. We conclude that TG-catalyzed Relish cross-linking suppressed the IMD signaling pathway to enable immune tolerance against commensal microbes.